人单核细胞泡沫化敏感候选基因的筛选

A Gene in Foam Cell Formation in Monocyte-Derived Macrophages Induced by ox-LDL with Suppression Subtractive Hybridization

为克隆调控单核细胞源性泡沫细胞形成的相关基因 ,采用抑制消减杂交法筛选U937细胞经氧化型低密度脂蛋白温育形成泡沫细胞后差异表达的基因。经过正向、反向两轮消减杂交和巢式聚合酶链反应扩增 ,获得了富集的差异表达的cDNA片段 ,即表达序列标签 ,克隆化后挑选经鉴定含有插入片段的质粒测序。经Genbank数据库进行同源比较 ,获得 2 0余个差异表达的EST ,其中 2个克隆FRG4和FRG1 4只有片段同源序列而无全长同源序列 ,提示可能来自新基因。Genbank登录号为 :FRG4(BI 50 2 586)和FRG1 4 (BI 50 2 587)。

Aim Identification of genes that differentially expressed during foam cell formation is important for understanding the molecular basis of atherosclerosis. Methods We used suppression subtractive hybridization(SSH) to isolate differentially expressed cDNA species in foam cells induced by incubation of U937 cells in the presence of oxidized LDL. This led to identification of more than twenty differential expressed sequence tags(EST) after further cloned and sequenced and homology searched in Genbank with software advanced BLAST 2.0. Results EST FRG4(a 273 bp cDNA) and FRG14(a 346 bp cDNA) had very low homology with sequence in Genbank, which may be new genes. Moreover, the results from RT PCR confirmed that FRG4 and FRG14 are indeed genes induced by ox LDL. The sequences have been assigned the database accession numbers in Genbank as below: BI50286, BI502587. Also we have found other genes such as human ubiquitin proteasome pathway and human retinoblastoma binding protein 4 differentially expressed in foam cells. Conclusions Suppression subtractive hybridization(SSH) is a kind of high efficient method to clone differentially displayed genes in tester and driver. We have found genes or ESTs may be related genes of atherosclerosis.